Author: Bhodi Tims

Although cannabis in the marketplace is not sourced from “wild grown” material, as are several important herbal supplements, there are factors associated with ecological systems influencing secondary metabolite profiles. Chemovar status has the greatest significance on the phytochemical makeup of cannabis, which is greatly impacted by the plant genetics. Small et al. (2016) noted that the recent phenomena of high THC strains show a heterogeneity of secretory gland head size, based on selection pressure that reflects genetically unstable plant populations. Along with the genetics, the private knowledge of grower or producer also has tremendous effect on the product.

This blog post will explore ramifications associated with production parameters, physiological stress, and elicitor effects. See this previous post that focused more exclusively on light and water effects on secondary metabolites.

Backer et al. (2019) reported that several elements increased the THC levels – increased plant density, optimal temperature and application of plant stress hormone ABA. The light intensity also greatly influenced the concentration of THC and CBD per meter squared. Based on review of existing literature the optimal light spectrum, intensity and duration has not yet fully elucidated, except perhaps by specific growers and held as private knowledge. Until this is more broadly known, modeling the various influences will be difficult. An additional piece of data that would be worthwhile is whether multiple influences act in a linear or more complex fashion to alter the makeup of cannabinoids and terpenes.

A more recent innovation, LED lighting, displays greater spectral elasticity and lack the heat associated with more traditional High-Pressure Sodium Vapor (HPS) lights. Bilodeau, et al. (2019) described the influence of different light regimens. They provided additional support for the strategy of short blue light photoperiod (12/12) to increase cannabinoid levels. They found that red light treatment increased number of roots/plant, flowering quantity and duration. Although no association between root and flower numbers has been elucidated, I’m curious to see mycorrhizal inoculation and red-light exposure combined might increase cannabinoids and/or terpenes. Ecological influences are rarely a single element but mimicking complex inputs can be challenging to model statistically.

Magagnini et al. (2018) combined blue light and UV-A to increase levels of CBG. Previously, Lydon, et al. (1987) had reported that UV-B exposure increased THC concentration, that CBD was more labile to UV-B exposure than either THC and CBC. This points to opportunities to use lighting strategies for altering the phytochemical profile to match clinical needs focused on minor cannabinoids.

Blue light dwarfing is a known industry phenomenon leading to a plant habitat more horizontal than vertical as found in cannabis plants grown in full spectrum lighting. Researchers were able to increase total cannabinoid content by 66% using blue regimen compared with HPS lights (Namdar, et al., 2019). Is there a useful trade off of plant size to gain increased cannabinoid concentration that might be influenced by new harvesting strategies?

Harvesting strategies in general are underappreciated as a quality tool. In reporting the agricultural practices at GW Pharmaceuticals, Potter (2014) noted that kin function within populations strategically allocated primary and secondary metabolite synthesis based on environmental stressors. This suggests a sampling strategy to normalize the variation in secondary metabolites.

In an unsuccessfully attempt to use leaf cannabinoid content to predict floral levels, Richins et al. (2018) did discover that if chemovar was THC dominant, there was almost no CBD in the leaf; while in CBD dominant chemovars, at least 0.5% CBD was found in the leaf. They also characterized several high THC Canadian and Dutch strains, finding that those chemovars were associated with predominant terpenes b-myrcene, a- and b-caryophollene, terpenoline and d-limone. The different strains were not sourced from the same chemovar heritage, so the similarity might be driven by the selection for high THC content.

Beyond light influences, Caplan et al., 2019 reported drought stress effect increasing terpene content in following medicinal plants Melissa officinalis, Nepata cataria, and Salvia officinalis. I imagine similar changes to terpenoid profiles might be gained in cannabis. Often noted for its aromatic quality, many of the terpenes produced by cannabis are known to possess insect-repellent properties. Among these are alpha and beta pinene, limonene, terpineol and borneol. Hood et al. (1973) noted that relative concentrations of terpenes with cannabis trichomes differed substantially from the volatiles detected in the atmosphere surrounding the plant. This suggests that specific terpenes maybe released in response to herbivory, which should alter the terpenoid profile that remain stored in the trichomes. As a strategy could a specific ration of these compounds be generated that has specific therapeutic properties?

If we approach altering the secondary metabolite through soil influences, it’s unclear whether inoculating cannabis plants with mycorrhizal fungi will do much to increase the secondary metabolite cannabinoids and terpenes. Pate’s review (1994) of the chemical ecology of cannabis provided evidence that soil potassium was negatively correlated with the cannabinoid concentration, suggesting that mycorrhizal association might reduce D-9-THC levels. Mycorrhizal fungi extend the reach of plant root system’s access to soil K in exchange for soluble sugars produced by the plant during photosynthesis.

Plant morphological change also mean cannabinoid ratios can be altered depending on the phase of plant growth. This becomes important in producing plants with more dominant ratios of specific cannabinoids. de Meijer et al. (2003) showed that in early growth stages the precursor CBG is limited, but that of the cannabinoids produced downstream (THC, CBC and CBD), CBC is most efficient during early growth, thus increasing its relative ratio. As the availability of CBG increases so does the relative concentration of THC and CBD. At late stages CBG levels are substantially reduced.

Finally, looking at micropropagation studies to characterize their impact on secondary metabolites Kodyn and Leeb (2019) reported on a reliable method to produce cannabis plantlets of excellent quality, requiring no added sugars, vitamins or plant growth regulators that limits microbial contamination and preserve genetic stability of the stock. A next investigation should look at the effects on the secondary metabolite profile and potentially produce changed cannabinoid and terpenes.

As the industry continues to stress growth of quality and standardization, understanding the chemical ecological influences on the actives fractions found in cannabis will become increasingly important.

As the FDA progresses toward a regulatory framework for hemp based cannabinoid products, I’ll touch on some unique research from the production side, focusing on lighting and hydration strategies in greenhouse settings and provide examples of how formulary might help drive the selection. Look for discussions on extraction methods or strain/chemovar choice at another time.

At a 2019 Cannabis Science Conference East talk, typical production methods for cannabis production were described as follows (Kern, 2019):

High light intensity and CO2 concentration

Photoperiod

Vegetative growth at 18 hours

Flowering < 12 hours

Use of Blue light during flowering

The blue light treatment is used to optimize several factors:

Increase terpenes with all blue light last three days of flower development

Blue light at end of day can increase leaf expansion increasing photosynthesis and plant mass yield

Research by Mosaleeyanon et al. (2005) showed that light impacts secondary metabolite production, with increased photosynthetic rates leading to increased total hypericin (hypericin + pseudohypericin) concentration in St. Johnswort.

Hawley et al. (2018) experimented with the use of subcanopy lighting (SCL) of red/blue (“Red-Blue”) or red-green-blue (“RGB”) light versus no SCL in a greenhouse production cannabis. They used a plant layout design to limit the amount of additional radiation from the subcanopy and to prevent the treatment light from influencing other plants. They ran the experiment without gyping (removal of the bottom 20 cm of stems). The SCL treatment created differences in the lower canopy metabolite concentrations:

From a QA perspective, the RB SCL treatment also provided the most consistent levels of cannabinoids and terpenes in both upper and lower canopy.

Hawely et al. (2018) suggested previous research (Miller et al., 1995: Zur et al., 2000) had demonstrated light spectra rich in green light was largely absorbed by terpenes, and that the plant had to up regulate the biosynthetic precursors in response to increased green light stimulus. The precursor molecules turn out to be part of both terpene and cannabinoid metabolic pathways, leading to an enriched biosynthetic stream. Greater precursors available led to increased production of both class of metabolites

Two examples of how formulary might take advantage of ecological influences

CBC and CBG concentrations have been shown to be present in leaves at equal or greater levels than in the flowers (Bernstein, Gorelick and Koch, 2019). This suggests that lighting and harvesting strategies may include adding leaf material to an extract and/or optimizing the impact of different light spectra on leaf metabolite profiles.Taking it a step further, cold water extraction of immature leaf of selectively bred cannabis chemotypes yields enriched CBC fraction (Potter, 2009)

Since CBC is the second most prevalent cannabinoid compound found in cannabis (Russo 2011), and research has shown topical anti-inflammatory activity (De Petrocellis et al., 2012: Cascio & Pertwee 2014; Oláh et al., 2016), and sebum reduction in acne, then leaf material might be combined with flowers in an extract. And the impact of under canopy lighting should also be investigated more extensively to determine if the leaf yield of cannabinoids and terpenes can be improved. Applying the RBC-SCL strategy to increase terpenes, the following enriched fractions might improve the results as an anti-acne topical: alpha-pinene as an effective antibacterial (Appendino et al., 2008); limonene, pinene and linalool decreasing sebum/sebocytes (Biro et al., 2009).

Experimenting with the yield in various chemovars based on multiple ecological inputs will make for a challenging project. The use of ecological inputs as one strategy for creating treatment specific strains might eventually be combined with chemovar selection and extraction protocols to create a highly specific therapeutic product.

The joy of moment has always been a huge part of my life. I was a hiding go seek champion at a young age, frustrating my elder siblings and cousins; I started out boxing at 11 years old. I added soccer and martial arts in middle and high school respectively, continuing to explore my love of movement. I found expression for my own darkness with combat martial arts in the ring and on the street. And somewhere during that time I fell in love with modern dance. I studied for several years at George Mason University, but in the end moved into a more predictable career path.

We move thru the world carrying dark and light in varying ratios, depending on life circumstances and choices we make. Of late I’ve found ways to immerse myself in my own darkness as a creative act as opposed to an embrace of violence – dancing the blues, or surprisingly, lifting weights. The challenge of pushing beyond my preconceived limits requires some letting go, and the companionship of that darkness is welcome at the edge.

So I’ll leave you with a blues number that has been my partner in varying guises of late and hope it finds you smiling and wanting to move!

The theory of evolutionary radiation of flowering plants has been linked to interaction with pollinators and herbivores (Ehrlich and Raven, 1964: Herrera et al., 2002). One class of phytochemicals that researchers investigated to understand the mechanisms for a co-evolutionary impact is pyrrolizidine alkaloids (PAs). Much of the past research has been on the toxicity of these compounds in various structural iterations. When they contain 1,2 double-bond (unsaturated) in their base, necine moiety (Figure 1), PAs can be activated to become hepatotoxic, carcinogenic, genotoxic and teratogenic to humans (IPCS 1988).

Symphytum officinale

This toxicity impacted the dietary supplement market in 2001, when the USA FDA required withdrawal of PA-containing Comfrey (Symphytum officinalis) preparations from the market. Nevertheless, other organisms have benefited from ingesting the compounds.

Biological data revealed how insects interactions with PA containing plants can be protective to the insect (Berenbaum and Aangerl, 1993; Nelson and Kursar, 1999). More recently, Liu, Vrieling and Klinkhamer (2018) explored these synergistic effects with herbivores. Their study evaluated the anti-herbivory effect of PAs on the study insects, Western Flower Trips (WFT). They found that plant secondary metabolite fractions underestimates the effect on herbivory. All fractions contained N-oxides and free bases. Most of the anti-herbivory effect was restored when fractions were recombined.

The insects were more susceptible to the more polar fraction of butanol than the chloroform fraction. Both N-oxides and free bases were present in higher concentration in the butanol as opposed to the chloroform, but total PA amounts were 3x

greater in later. They also noted that when combined with chlorogenic acid (CGA), the N-oxide was more active than when combined with free base, which is the opposite of what was expect based on toxicity profiles.

One of the more important findings from their study was that aside from the N-oxide and freebase forms of the PA, other metabolites can act as antagonists or synergists between CGA and the PA retrorsine. The complexity of the phytochemical background altered the interactions between plant metabolites and their potential bioactivity. What we don’t know is if the use of laboratory spiking studies will work in the field, and will it have population level effects necessary for long term alterations to behavior?

Another piece of interesting research focused on where these compounds get sequestered in the plant. Stegemann et al. (2018) investigated the accumulation and role of PAs’ in comfrey (Symphytum officinale) flowers and fruits. It was originally believed that PA synthesis occurred in the plant roots, which was then distributed to the rest of the structure. They reported secondary sites of synthesis in young leaves subtending developing inflorescences, with transport from leaf to flower to protect reproductive structures.

The authors found variability in accumulation patterns in different tissue, strongly suggesting that the synthesis is developmentally driven. Generally, the level of N-oxides to tertiary PAs was ~95% in all tissue , and the fresh weight PA concentration (ppm) present in different plant parts are listed as follows (from least to most):

sepals (tr)

petals (6)

pollen (14)

ovaries (98)

complete flower (141)

fruits (183)

The patterns of the alkaloids themselves were different in various tissue. Application of methyl jasmonate didn’t appear to alter the levels of expression, thus they appear not to be constitutively produced. The highest levels appeared at peak inflorescence, dropping as the flowers withered. And additional PAs are stored in young leaves and reproductive structures.

In table below, even though only trace amounts of all PAs are located in petals and pollen, almost all is the PA is myosorpine. It’s not clear why that pattern of individual PAs is unique to specific tissue. The presence of specific PAs in different tissues may be “leftover” from the original metabolic pathway, with the plant shuttling those particular metabolites to specific tissue.

7-acetylintermedine

~65%

ovaries

fruits

whole flower

~45%

leaves

roots

7-acetyllycopsamine

~30%

leaves

roots

intermedine

~15% all

lycopsamine

~15%

ovaries

fruits

whole flowers

5%

leaves

12%

roots

myosorpine

99%

petals

pollen

3-acetylmyoscorpine

largely trace

We can compare these unique distribution and accumulation patterns to plants that normally don’t produce PAs. Now et al. (2016) tested horizontal transfer of natural products from leachate of rotting plants containing PAs into plants that do not produce those compounds. The accumulation of PA’s occurred in the leaves of guest plant, not flowers, suggesting xylem transfer driven via transpiration. Accumulation in flowers via transport (as opposed to being a production site) is typically source-sink-translocation via phloem. The researchers found that the PAs accumulated as salt like N-oxides. They had expected the presence of free base PAs since they are able to cross biomembranes via simple diffusion. Either the guest plant has a transporter able to translocate N-oxides, or the free base PAs are taken up and oxidized to N-oxide. It made me wonder if this root uptake of exogenous PA’s might happen in all plant species, or was it limited specific plant species, genus or families?

Finally, Wink (2019) discussed how PAs are often stored by specialized insects and that their chemical ecology is intricate. As an example Wink noted how they function as nuptial gift for defense of eggs in the caterpillar larvae of Creatonotus spp. after metamorphosis into adult insects. The presence of the PAs secreted on to the eggs protects them from potential predators. Because of that benefit, it appears that increased PA ingestion by male caterpillars enhances their attractiveness.

This last puzzle in the world of PA chemical ecology had me wondering what examples might exist in the human world that mimic this type of behavior. Although beer consumption has long been thought to enhance the appearance of human male virility, my own experience has disproved that particular absurdity. Do you have any examples?

In their final trimester, Masters students in the Herbal Product Design and Manufacture Program at Maryland University of Integrative Health (MUIH) take a case study course where companies and various stakeholders present real world problems for our students to solve. The current iteration will investigate the significant public interest in cannabis (hemp)-based and CBD-containing food and herbal products.

Many questions were generated about the science, safety, and quality of these products at the May 31, 2019 public hearing with the Food and Drug Administration. Key take-away messages from this meeting are the following:

Data are needed to determine safety thresholds for CBD.

Datasets/information should be objective, of adequate quality and available for transparent review.

Lab testing and data analyses need to be replicable.

Consumers need consistent information and labeling.

State/government entities need support in knowing what to do.

We need to understand the implications for children when they take CBD-containing products at different dosage ranges.

This has its own challenges. The FDA has identified several safety based-questions about CBD products that need answers as noted above. For example:

How much CBD is safe to consume in a day?

How does it vary depending on what form it’s taken?

Are there drug interactions that need to be monitored?

What are the impacts to special populations, like children, the elderly, and pregnant or lactating women?

What are the risks of long-term exposure?

Many industry insiders believe that hemp-based cannabis products, such as cannabis oil or tinctures will be addressed using the DSHEA statutory framework, but that these products will require New Dietary Ingredient (NDI) filings. A description of the 2016 NDI guidance document can be found at https://www.fda.gov/media/99538/download.

To that end, our case study this trimester students will collaborate with stakeholder Dr. Roger Clemens. He is internationally known scientist, and Professor of Pharmacology and Pharmaceutical Sciences and Assistant Professor of Regulatory Science within USC’s School of Pharmacy, International Center for Regulatory Science. Dr. Clemens is a past president of the Institute of Food Technologists (IFT) and is the current presiding officer of the International Academy of Food Science and Technology. He co-founded, established and contributes to a Food, Medicine and Health column published monthly in Food Technology for the past 15 years. He continues to serve on several editorial boards (e.g., Food Chemical Toxicology, Journal of Food Science, Toxicology Research and Applications, Journal of Dietary Supplements) and continues to serve as a reviewer for many high-impact journals. Dr. Clemens has published > 50 original manuscripts and commentaries. In addition, he has presented more than 250 invited lectures at domestic and international scientific conferences. He is an elected Fellow in four scientific organizations.

They will be tasked with designing an effective model for a hemp oil NDI (New Dietary Ingredient), which will serve them in two important ways: 1) acquire a deeper understanding of hemp constituents and their safety (classic ADME and toxicology), public health implications, and potential medicinal application; and 2) learn to write an NDI, which is vital in the herbal supplement industry.

Plants make numerous small molecules (metabolites) that are either directly toxic to insects, grazing animals, fungi and bacteria, or that stimulate the production of other toxic metabolites. The production of various types of metabolites such as alkaloids, terpenes, and phenolics can be turned on (induced) after damage to the plant occurs. The control mechanisms involve jasmonate, salicylate, phytohormone (plant hormone) ethylene, the volatile (gaseous) methyljasmonate and methylsalicylate signaling pathways.

The signal process is complex. It works on a local level or systemically by traveling throughout the entire plant. It behaves like a network, allowing chemical communication between parts of a plant or within a population of plants in a given locale. With recent developments in metabolic profiling, highly sensitive separation and detection systems are used to create metabolite profiles, high-throughput gene expression analysis is used to detect genes transcripts and rigorous statistical mining resulted in some interesting data that reveals more about how plant defense signaling is controlled.

Researchers used metabolic profiling of overall patterns rather than relying on targeted metabolites. This is important, since previous work focused on a few major metabolites and provided conflicting data. They found differences in profiles from plants exposed to generalist insect feeders versus plants treated with phytohormones. Although both jasmonate and salicylate pathways were activated in each treatment (co-induced), the metabolite patterns were distinct; both treatments lead to a stronger localized rather than systemic response; and there appeared to be a great deal of cross-talk between both pathways influencing pools of precursor metabolites.

Botrytis cinerea growing on tomato leaf

Another study followed the spatial accumulation of hydroxycinnamates (phenylpropanoids) and lignins in cell walls of Arabidopsis (a model organism) in response to changes in the ethylene signaling induced by the necrotrophic fungal pathogen, Botrytis cinerea. They correlated metabolic profiles with cytological (cell based) changes to provide biological validation of the analytical data.

When a fungus like Botrytis attacks a plant, it generally destroys cells and eventually the entire plant. In the presence of the fungus, plant genes for the biosynthesis of phenylpropanoids and lignins are expressed (turned on) to modify and reinforce the plant cell wall against fungal penetration. Botrytis induces over 30 ethylene regulated transcription factors – cellular molecules that target and induce underlying genes to become active. So these researchers used a metabolite profile of 3 ethylene mutants, plants that had gene mutations at different DNA sequence points of the ethylene signaling pathway.

It turned out that the mutant plants were less resistant to the fungus and that ethylene resistance, when present, appeared after the fungus had made contact with the plant cell wall and had begun to build the structures necessary to penetrate the cell wall barrier. One phenylpropanoid metabolite in particular, ferulate, seemed to be highly influenced by ethylene signaling. Ferulate cross-links the polymer strands of cell wall polysaccharides, enhancing their structural integrity as a barrier.

So what does this have to do with good medicine? Don’t focus on one or two metabolites to make an efficacious extract. Despite what you hear, we are not trying to mimic “magic bullet” medicine. Expose plants to a full set of ecological challenges to produce a metabolite profile of greater diversity. Mono-crop farming doesn’t cut it. Damn if those hippies had it right after all.

This post also focuses on complex ecologies, found in education, not the wild. It reviews the adoption of an ePortfolio in our Therapeutic Herbalism Masters program at Maryland University of Integrative Health, which was designed to accomplish three main tasks:

Encourage and provide opportunities for our students to experience meta-cognitive learning about the competencies they have acquired/developed..

Create a professional web presence for the student to market their expertise.

Assess whether our program learning outcomes are being met in the classroom.

The Student Learning Portfolio allowed student to collect artifacts of their acquired competencies. These were often based on assignments from each of their courses. Ideally, they reflected skills developed, competencies, and career readiness resulting from each course and the integration of those experiences. The process was meant to enhance the ability of students to be self-sufficient reviewing their ability to succeed in a chosen profession.

Their final product was creation of a Professional ePortfolio, where the competency artifacts selected by a student showcased the knowledge, skills, and abilities that are in demand in the professional marketplace.

During our review of the tool it became apparent two important lynch pins were missing. Firstly was the articulation of appropriate professional competencies as measurable program learning outcomes. Secondly, our faculty had limited experience applying learning outcomes and reviewing meta-cognitive tools such as the ePortfolio. They were not prepared to highlight the connection between chosen artifact from their course, as well as the underlying learning process and how it was linked to an overriding professional competency.

A series of faculty retreats refined more effective and measurable program learning outcomes. In addition, the institutional assessment process that emerged out of a regular Middle States Higher Education Commission review of the university helped create more specific measurement goals. The combined effect enabled both faculty and students to identify appropriate artifacts and learning processes.

We articulated specific competencies that were missing or ill-defined in previous versions, including:

Improved research literacy skills – finding and assessing the validity of scientific research – in support of their analytical work on assignments.

Succinct summarization of primary, peer-reviewed resources and the synthesizing of new ideas from those summaries that contribute unique ideas to the field

For clinical students, populated their portfolio with case study write-ups.

Created effective narratives of how they worked with incomplete data (medicine making, research or diagnosis) in finding a solution using iterative problem solving.

Other challenges that appeared included the need to help students learn how to select learning artifacts that reflect project-based learning. Since the feedback loop in assessment provides data about how program outcomes are being met in the classroom, the process of student metacognitive review of both object and learning processes revealed difficulty in effectively linking the two.

Apparently this is not unusual in ePortfolio development (Land & Greene, 2000). At the core of this issue is the requirement for more engaged and knowledgeable faculty to embed assignments and course long assessment arcs focused on strengthening the linkage between an object (paper, case study, etc.…) and the underlying programmatic learning objective.

Faculty training in applying ePortfolio to their own professional development would improve their the ability to guide students into choosing suitable learning artifact and how to articulate those to employment marketplace.